NO151897B - PROCEDURE FOR PREPARING AN INSULIN OR AN INSULIN ANALOGUE - Google Patents

Description

The present invention relates to a method for producing an insulin or an insulin analogue.

With the possibilities that exist today to produce protein products using recombinant DNA methods and more particularly the production of the insulin A chain and the insulin B chain by the technique, which e.g. is described in Goeddel et al., Proe. Night'1. Acad. Pollock. USA 76, 106-110 (1979), there has been an ever-increasing need for an efficient method which can combine the aforementioned A and B chains into insulin.

Previously known methods for production

of insulin by combining the A and B chains, used as starting materials the A and B chains in the form of their stable S-sulfonates. Typically, the A- and B-chain S-sulfonates,

either separately or together, reduced to d corresponding to the -6H compounds, usually using a large excess of a thiol reducing agent. The products were then isolated from the reducing medium, and if they were not reduced together,

brought together in an oxidizing medium, e.g. air, thereby obtaining a combination of the A and B chains and a production of insulin. Examples of this method technique can i.a.

found in Du et al., Scientia Sinica 10, 84-104 (1961), Wilson et al., Biochim. Biophys. Acta 62, 483-489 (1962), Du et al., Scientia Sinica 14, 229-236 (1965), Kung et al., Scientia Sinica 15, 544-561 (1966), Kexue Tongbao (China) 17, 241 -277

(1966), and Markussen, J. Acta Paediatrica Scandinavica,

Suppl. 270, 121-126 (1977).

A modification of this method includes

that the A-chain S-sulfonate is reduced followed by a reaction between the reduced A-chain and the B-chain S-sulfonate in an oxidizing atmosphere. See e.g. Katsoyannis et al., Proe.

Nat. Acad. Pollock. (U.S.A.) 55, 1554-1561 (1966), Katsoyannis, Science 154', 1509-1514 (1966), Katsoyannis et al., Bio-chemistry 6, 2642-2655 (1967), U.S. Patent No. 3,420,810 and Jentsch, Journal of Chromotography 76, 167-174 (1973).

Another modification includes partial oxidation

tion of the A-chain SH bond to a disulfide between A-6-

and the A-II cysteine residues followed by an oxidation of the product

ted with B-chain SH- or B-chain S-sulfonate. See e.g.

Belgian Patent No. 676,069 and Zahn et al., Liebigs Ann. Chem. 691, 225-231 (1966).

In all of the above known methods it is

one common feature, i.e. the production of insulin by two independent successive steps, i.e. reduction of S-sulfonate to -SH, followed by oxidation to -S-S-.

Dixon et al., Nature 188, 721-724 (1960) describe conditions suggesting a simple solution conversion of A- and B-chain S-sulfonates to insulin using a thiol reducing agent and air oxidation. The details are relatively sparse, and the yield, based on the activity of the recovered product, is stated at 1-2%. However, Dixon is in Proe. Intern. Congress Endecrinol, 2nd ed., London 1964, 1207-1215 (1965) in more detail, suggesting in Table IV on page 1211 that the conditions indicated in the earlier publication involve separate reduction and oxidation steps.

In contrast to previously known methods

it has now been discovered that it is possible under defined reaction conditions to achieve satisfactory levels of

approach to the production of insulins or analogues of insulins from S-sulfonated A and B chains, by carrying out both reduction and oxidation reactions in a single step in a process where a single solution is used. The invention relates to such a method.

According to the present invention, it is thus provided

a method for producing an insulin or an insulin analogue by combining an A chain of an insulin or an insulin analogue and a B chain of an insulin or an insulin analogue, and this method is characterized by reacting the S-sulfonated form of the A-chain, the S-sulfonated form of the B-chain and a thiol reducing agent in an aqueous medium under such conditions that a mixture is obtained with (1) pH 8-12, (2) a total protein concentration of 0.1- 50 mg per ml and (3) a total amount of said

thiol reducing agent so that from 0.4 to 2.5 -SH groups per -SS03 groups in the total amount of said A- and B-chain S-sulfonates, whereby an insulin or an insulin analogue is formed by keeping the mixture at a temperature of 0-25°C in an environment that provides an oxygen source .

This method represents an efficient, single-step, single-solution process for the manufacture of insu-

lin or an analog of insulin from its S-suiphonated A and B chains.

The term "insulin" naturally means any naturally occurring insulin, such as that which occurs in humans, pigs, poultry, sheep, fish, birds etc., so-

as well as hybrids formed by a combination of an A chain from one species and a .B chain from another.

The term "insulin analogue" means any of

a number of proteins each containing the basic A-chain and B-chain containing all the half-cysteine residues

in the same order as found in natural insulins.

These analogues differ from natural insulins by substitution, addition, exclusion or modification in one or more amino acid residues, but have retained the disulfide bond arrangement and at least part of an insulin-like activity. Examples of such analogues are [N-formyl-Gly^"-A] insulin, Desamino-A^-insulin, [Sarcosin^-A]-1 21

insulin, [L-Alanine -A]insulin, [D-Aspargine -A]insulin,

21 0 21 21 [Isoaspargine -A]insulin, [D-Aspargine -A]insulin, [Arginine 21 1

A]insulin, [Asparaginamide -A]insulin, [Sarcosine -A, Asparagin 21 -A]insulin, [Norleucine 2 -A]insulin, [Threonine 5-A]insulin,

5 19 19 [Leucine -A]insulin, [Phenylalanine -A]insulin, [D-Tyrosine A]insulin, [Tyrosine 18 -A, Asparagine 19 -A, Arginine <21->A]insulin, 28—30 27— 30 Des[B -tripeptide]insulin, Des[B -tetrapeptide]insulin

lin, Des[B -pentapeptide]insulin, Des[B ~ -tetrapeptide, Tyrosinamide -B]insulin, Des[B ~ -pentapeptide, Phenylalanine amide^-B] insulin, [Leucine^-B] insulin, Des [Phenylalanine ^-B]-insulin and the like. These and other analogues are described in the literature, see e.g. Blundell, T. et al., Advances in Protein Chemistry, 26, 330-362, Academic Press, New York,

(1972), Katsoyannis, P.G., Treatment of Early Diabetes, 319-327, Plenum Publishing Corp. (1979), Geiger, R., Chemiker Zeitung, Reprint 100, 111-129, Dr. A. Huthig, Publsher, Heidelberg, West Germany (1976), Brandenburg, D. et al., Biochem. J. 125, 51-52 (1971).

Although the present method is applicable

for the production of insulins and insulin analogues, it is preferred to use it for the production of naturally occurring insulins, then especially insulin from humans, cattle or pigs, and then especially insulin for humans.

When carrying out the present method, a combination of the A and B chain for the production of insulin or an insulin analogue can be achieved within a wide range with respect to the ratio of one chain to the other. The combination is of course limited by the chain, whether this is A or B, which is present in the smallest quantity. In any case, although it is not critical, the usual ratio between the A and B chain on a weight basis will be from 0.1:1 to approx. 10:1. It is preferred to carry out the present method by using a weight ratio between the A and B chain in the range from 1:1 to 3:1. It has also been discovered that within this area there are certain areas which are particularly advantageous for the production of a particular type of insulin. When you e.g. want to combine an A and B chain for the production of bovine insulin, then it is preferred that the ratio between the A and B chain lies in the range from about 1.4:1 to 1.8:1. • With regard to porcine insulin, so

it is preferred that said range of variation is from 1.0:1.0 to 1.4:1.0. With regard to human insulin, so

is the preferred area from approx. 1.8:1.0 to approx. 2.2:1.0.

Another parameter that is important for carrying out the present method is the protein concentration in the reaction medium. The method can be carried out with good results within a wide range with regard to protein concentrations. Usually, however, the protein concentration will vary from 0.1 - 50 mg per ml reaction medium. Preferably, said concentration of protein should be in the range from approx. 2 - approx. 20 mg per ml. Again, it has been discovered that within the aforementioned range of variation there are optimal protein concentrations that vary depending on the type of insulin that is produced. If you e.g. wish to produce pig insulin, it is preferred that the protein concentration varies from approx. 8 - 16 mg per ml, while in the production of human or bovine insulin there is a preferred range from approx. 3 - approx. 8 mg per ml.

The method according to the present invention is carried out in an aqueous medium. The pH of said medium, measured at room temperature, will usually range from approx. 8 - approx. 12. Preferably it will lie in the range from 9.5 - 11.0 and optimally in the range from approx. 10.4 - approx. 10.6. The pH in the medium can be maintained in the desired range by means of suitable buffering agents. Typical buffering means in this respect are, for example, glycine, glycylglycine, carbonate, tris(hydroxymethyl)aminomethane, N,N-bis(2-hydroxyethyl)glycine, pyrophosphate, fil-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid and the like which effectively provide a pH control within the above-mentioned areas. A common and preferred buffering agent is glycine.

The concentration of the buffering agent usually ranges from approx. 0.001-molar to 2-molar. The preferred concentration is from 0.01 to approx. 1 molar, most preferably from 0.01 molar to 0.1 molar.

The A and B chains are brought together in said suitable aqueous medium in the presence of a thiol reducing agent. The term "thiol reducing agent" means a compound which contains at least one -SH group and which has the ability to effectively reduce the S-sulfonate groups in the A and B chains. Although any compound having the aforementioned properties can be used, it is preferred that the thiol reducing agent used is one which, in its oxidized form, can be cyclized to a very stable compound. The thiol reducing agent is used in an amount that gives from 0.4 - 2.5 -SH groups per -SS03~ groups which are present in total in the A and B groups, and preferably the amount should be such that you get 0.9 - 1.1 -SH groups per each

-SSO^- group.

Examples of typical thiol reducing agents are dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, methylthioglycolate, 3-mercapto-1,2-propanediol, 3-mercapto-propionic acid, thioglycolic acid, 2-amino-3-mercaptopropionic acid (cysteine ) and other such thiol compounds. Preferred thiol reducing agents are dithiothreitol and dithioerythritol and of these dithiothreitol is the most preferred.

An essential feature of the present method is that it is carried out in an environment that provides an oxygen source. This can easily be achieved by exposing the reaction mixture to air. Although a more direct contact method can be used, e.g. by bubbling air or oxygen into and through the reaction medium, this is usually not necessary.

The present method is therefore generally carried out by preparing a mixture of A-chain S-sulfonate, B-chain S-sulfonate and said thiol reducing agent in the desired concentrations in an aqueous medium at a pH of 8-12. The mixture, which is open to air contact, is carefully stirred for a sufficiently long period of time to obtain a chain combination, i.e. usually at least approx. half hour. During this stirring period, the mixture will generally be kept at a temperature of approx. 0 - approx. 25°C, however, it is usually preferred that the mixture is subjected to moderate cooling so that the temperature remains at the lower end of said temperature range, usually from approx. 2 - approx. 10°C.

As soon as the reaction is complete, insulin or the insulin analogue product can be isolated in a number of different ways, and a number of these are known within the insulin technology. The most commonly used technique for insulin purification is the chromatographic technique. These can easily be used for the recovery of insulin produced by means of the present invention. Said technique may include gel filtration chromatography or ion exchange chromatography. Furthermore, the product can be assessed for purity and activity using known methods, such as polyacrylamide gel electrophoresis, amino acid analysis, insulin radioreceptor test, insulin radioimmunotest, high pressure liquid chromatography (HPLC), ultraviolet spectrum examination, dansylation, tests using rabbit blood glucose and the like.

The insulins available by the present method include hybrids consisting of the A chain from one type of insulin and the B chain from another type. The present invention generally relates to a combination of the A- and B-chain S-sulfonates, and the special structure that may exist in these chains, as long as they are real representatives of insulin and insulin analog A- and B-chains are without significance for the present method.

Although one can prepare an insulin analogue or an insulin hybrid, e.g. using an A chain from one type of insulin and a B chain from another, it is of course preferred to produce an insulin that is structurally identical to a naturally occurring insulin, which can be done by using an A -chain S-sulfonate and a B-chain S-sulfonate, each of which has the same amino acid sequence as in said natural insulin. Furthermore, it is highly preferred to use the present method to produce pig insulin, bovine insulin or human insulin, and it is most preferred to produce the latter type of insulin.

Insulin or insulin analog A or B chains are, as already indicated, accessible by means of recombinant DNA probes. They can also be produced from natural insulins or by classic peptide synthesis techniques, whether this takes place in solid phase or in solution.

The A and B chains are kept in stable form as S-sulfonates. These S-sulfonate starting materials are available by oxidative sulfitolysis, a treatment by which the A and B chains are reacted with sodium sulfite in the presence of a weak oxidizing agent, such as sodium tetrathionate.

The following examples illustrate the invention. The examples are provided for illustrative purposes only and are thus not limiting of the invention as such.

Example 1

360 mg porcine A-chain S-sulfonate was dissolved in

36 ml of 0.1-molar glycine buffer (pH 10.5) and the pH of the mixture was adjusted to 10.5 with 5N NaOH. Porcine B-chain S-sulfonate (300 mg) was dissolved in 30 ml of 0.1-molar glycine buffer (pH 10.5) and the pH of this mixture was adjusted to 10.5 with 5N NaOH. Dithiothreitol (DTT) (123.4 mg) was dissolved in 0.4 ml of said 0.1-molar glycine buffer (pH 10.5) and the pH of the mixture was adjusted to 10.5 with 0.2 ml of 5N NaOH. The A and B chain solutions were combined in a 100 ml vial at room temperature (about 25°C) and 1.91 ml of DTT solution was added to give a -SH to -SSO^ ratio of 1.04 . The resulting solution was gently stirred in an open beaker with magnetic stirring at 4-8°C for 20 hours. Analysis by high pressure liquid chromatography (HPLC) indicated an insulin yield of 193.8 mg or 29% of the total protein. 40 ml of this solution was adjusted to pH 3.15 with acetic acid. The mixture was gel filtered on a 5 x 200 cm column of "Sephadex G-50" (Superfine) equilibrated and eluted with 1-molar acetic acid at 4-8°C. The insulin peak (elution volume 2450-2700 ml) was combined and lyophilized with a yield of 95 mg insulin or approx. 25% of the total protein. Said porcine insulin was judged to be relatively pure by a polyacrylamide gel electrophoresis, amino acid analysis, insulin radioreceptor assay and HPLC and a rabbit blood glucose reduction assay.

Example 2

Solutions of bovine A- and B-chain S-sulfonates with a concentration of 5 mg per ml in 0.01-molar glycine buffer (pH 10.5) was prepared. The pH of each of the solutions was adjusted to 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved

in 4.0 ml of 0.1-molar glycine buffer (pH 10.5) and the pH was adjusted to the mentioned value with 0.15 ml of 5N NaOH. 0.5 ml of the B-chain solution was added to 0.8 ml of the A-chain solution and 0.035 ml of the DTT solution at room temperature (approx. 25°C), whereby a -SH to -SS03~ ratio of 0.91. The result-

the final solution was stirred at 4 - 8°C for 20 hours in a 3 ml ampoule. High pressure chromatography analysis of the mixture indicated a yield of bovine insulin of 1.96 mg or approx. 30% of the total protein.

Example 3

Solutions of porcine A- and B-chain S-sulfonates with a concentration of 10 mg per ml in 0.1-molar glycine buffer (pH 10.5) was prepared. The pH of each solution was adjusted to 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved in 2.0 ml of glass distilled water. 0.5 ml of the B-chain solution was added to 0.6 ml of the A-chain solution, and the whole was added to 29.25 µl of the DTT solution at room temperature (about 25°C), whereby a -SH to - SS03" ratio of 1.00. The resulting solution was stirred at 4-8°C in an open 3 ml vial for 20 hours. A HPLC analysis of the mixture indicated a yield of porcine insulin of 3.81 mg or about 35% of the total protein.

Example 4

Human (pancreatic) B-chain S-sulfonate and several types of human (pancreatic and E. coli) and porcine (pancreatic) A-chain S-sulfonate solutions were prepared with a concentration of 5 mg per ml in 0.1-molar glycine buffer

(pH 10.5). The pH of each solution was adjusted to 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved in 4.0 ml of 0.1-molar glycine buffer (pH 10.5), and the pH adjusted to this value with 0.16 ml of 5N NaOH. 1.0 ml of each of the A-chain S-sulfonate solutions was added to 0.5 ml of the B-chain S-sulfonate solution and 0.05 ml of DTT solution at room temperature (approx. 25°C), whereby a -SH to -SS03~ ratio of 1.09. All solutions were stirred at 4 - 8°C in an open ampoule for 20 - 22 hours. They were then analyzed by means of high-pressure chromatography using a human pancreatic insulin standard to calculate the yield. The results are indicated in the table below.

Example 5

A solution of each of the human A- and B-chain S-sulfonates was prepared at a concentration of 5 mg per ml in 0.1-molar glycine buffer (pH 10.5). The pH of each solution was adjusted to 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved in 4.0 ml of 0.1-molar glycine buffer (pH 10.5),

and the pH was adjusted to 10.5 with 0.16 mL of 5N NaOH. 0.5 ml of said B-chain solution was added at room temperature to 1.0

ml A-chain solution followed by 50 µl of the DTT solution to give a -SH to -SS03~ ratio of 1.09. The resulting solution was stirred at 4-8°C in an open 3 ml vial for 22 hours after which analysis by high pressure chromatography indicated a yield of human insulin of 2.58 mg or 34% of the total protein.

Example 6

328 mg of human A-chain S-sulfonate was dissolved in

65.6 ml of 0.1-molar glycine buffer (pH 10.5), and the pH of the mixture was adjusted to 10.5 with 75 µl of 5N NaOH. 164 mg of human B-chain S-sulfonate was dissolved in 32.8 ml of 0.1-molar glycine buffer (pH 10.5), and the pH of the mixture was adjusted to 10.5 with 15 ml of 5N NaOH. 61.7 mg of DTT was dissolved in 4.0 ml of said 0.1-molar glycine buffer (pH 10.5) and the pH of the solution was adjusted to said 10.5 with 160 µl of 5N NaOH.

The A and B chain solutions were combined in a 150 ml beaker at room temperature (about 25°C) and 3.28 ml of DTT solution was added to give a -SH to -SS03~ ratio of 1.09 . The open beaker was placed in an ice-water bath in a cooled room and stirred vigorously for half an hour. The solution was then stirred for a further 24 hours in a cooled room at 4 - 8°C. Chromatographic analysis at this time indicated a yield of human insulin of 148 mg or 30% of the total protein.

100 ml of this solution was added to 25 ml of glacial acetic acid to a final pH of 3.15. This entire sample was gel filtered on a 5 x 200 cm column of "Sephadex G-50" (Superfine) equilibrated and eluted with 1-molar acetic acid at 4-8°C. All eluted protein was lyophilized. The insulin peak (elution volume 2465-2781 ml) weighed 125 mg and represented 29.4% of the recovered protein.

Part of the above insulin peak (95.5 mg)

was dissolved in 9 ml of 0.01-molar Tris-0.001-molar EDTA - 7.5-molar urea-0.03-molar NaCl buffer (pH 8.5 at 4°C). The mixture was chromatographed through a 2.5 x 90 cm DEAE (diethylaminoethyl) cellulose ion exchange column equilibrated with the same buffer. The protein was eluted at 4-8°C with a gradient of 1 liter each of 0.03-molar and 0.09-molar NaCl in the same buffer followed by 1 liter of buffer containing 1-molar NaCl. Each of the peaks was desalted on "Sephadex G-25"

(coarse type) column in 2% acetic acid and lyophilized. The insulin peak (elution volume 878-1008 ml) weighed 55.73 mg and represented 84% of the recovered protein.

Zinc insulin crystals were prepared by dissolving 11.90 mg of the insulin (DEAE) peak sample in 240 µl of 0.1N HCl followed rapidly by 2.16 mL of a 0.04% solution of a ZnC^-0.05-molar sodium citrate-15 % acetone in a glass centrifuge tube. The crystallization takes place within 72 hours at room temperature (approx. 25°C), after which the overlying liquid was removed and the crystals were washed twice with cold water at pH 6.1 and then centrifuged at 2000 rpm. at 3°C between washings. The crystals were then redissolved in 0.01N HCl for analysis.

The resulting human insulin preparation was judged to be relatively pure by polyacrylamide gel electrophoresis (single band), amino acid analysis, insulin radioreceptor assay, insulin radioimmunoassay, HPLC, dansylation and

UV spectrum. The USP rabbit test (144 rabbits) gave a potency

of 26.3 - 1.8 units per mg (anhydrous).

Example 7

Solutions of human (E. coli) were prepared

[N-formyl-Gly^]-A-chain S-sulfonate and human (pancreatic) B-chain S-sulfonate in a concentration of 5 mg per ml in 0.1-molar glycine buffer (pH 10.5). The pH of each solution was adjusted to 10.5 with 5N NaOH. 61.7 mg of DTT was dissolved in 4.0 ml of 0.1-molar glycine buffer (pH 10.5), and the pH was adjusted to 10.5 with 0.16 ml of 5N NaOH. 0.5 ml of the B-chain S-sulfonate solution was added to 0.1 ml of the [N-formyl-Gly"'"]-A-chain S-sulfonate solution and 0.05 ml of the DTT solution at room temperature (25°C), whereby a -SH to -SSO^ ratio of 1.10 was obtained. The solution was stirred in a cooled room

(4 - 8°C) in an open 3 ml vial for 23 hours and an analysis indicated a yield of [N-formyl-Gly^-A] human insulin of 1.46 mg or 19.5% of the total protein.

After acidification to pH 3.15 with glacial acetic acid, a

part of the solution gel filtered on a 1.5 x 90 cm column of "Sephadex G-50" (Superfine) equilibrated and eluted with 1-molar acetic acid at 4 - 8°C. ■ The [N-formyl-Gly^A] human insulin peak (elution volume 87-95 ml) was pooled, aliquoted and lyophilized. The protein was judged to be relatively pure by chromatographic analysis and amino acid analysis. The bioactivity of this [N-formyl-Gly^-A] human insulin was judged by means of a radioreceptor test to be 17% in relation to a human insulin standard.

Example 8

A solution of porcine A-chain S-sulfonate and

human (E. coli) B-chain S-sulfonate was prepared at a concentration of 10 mg per ml in a 0.1-molar glycine buffer (pH 10.5). An A-B mixture was prepared by using 2 ml of the A chain solution for every 1 ml of the B chain solution. The A-B mixture was adjusted to pH 10.5 with 5N NaOH. 121.2

mg of cysteine was dissolved in 3.0 ml of 0.1-molar glycine buffer (pH 10.5), and the pH was adjusted to the mentioned value with 0.35 ml of 5N NaOH. 1.4 ml of the A-B mixture was added at room temperature

added 52 ul of the cysteine solution, so that a -SH to -SSO^ ratio of 0.95 was obtained. The resulting solution was stirred at 4 - 8°C in an open 3 ml vial for 20 hours and chromatographic analysis gave a human insulin yield of

3.25 g or 23.2% of the total protein.

Claims (1)

Method for producing an insulin or an insulin analog by combining an A chain of an insulin or an insulin analog and a B chain of an insulin or an insulin analog, characterized in that one reacts the S-sulfonated form of the A chain, the S-sulfonated form of B- the chain and a thiol reducing agent in an aqueous medium under such conditions that a mixture is obtained with (1) pH 8-12, (2) a total protein concentration of 0.1-50 mg per ml, and (3) a total amount of said thiol reducing agent so that 0.4 to 2.5 -SH groups per -SS03~ group in the total amount of said A- and B-chain S-sulfonates, whereby an insulin or an insulin analogue is formed by keeping the mixture at a temperature of 0-25°C in an environment which provides a oxygen source.